1. Field of the Invention
The present invention relates to methods for determination of a substance by measurement of light emitted from a specimen prepared based on an antigen-antibody reaction. In particular, the present invention relates to solid phase immunoassays by use of fine particulate materials.
2. Related Background Art
The antigen-antibody reaction is one of the reactions with a high affinity and high specificity. The immunoassay method using the antigen-antibody reaction allows to detect objects such as living tissues, cells, and bacteria, with high sensitivity. Further, the immunoassay method is simple in operation and applicable to various objects. Therefore, this method is widely used for measurements in biology and biochemistry. In recent years, various methods are being actively developed featuring such advantages, especially for ultrasensitive assays of trace substances; for example, interleukin 6 in human blood.
In conventional fine-particle solid phase immunoassay by light measurements, a point sensor is generally used to determine the intensity of signals from the sample. In enzyme immunoassays, for example, an enzyme activity is either transduced into fluorescent light or other emissions. And then the light can be detected by a fluorescence spectrophotomer and other instruments including a photomultiplier tube as a detector.
In point sensing with a point sensor, however, the measurement is affected by background noises emitted from the entire area of the sample's optical image. The noises typically come from contaminants in the sample. When the signal is weak in the point sensing, the sum of those noises across the image is much higher than the sum of the intensity of the signal from the object, providing a low S/N ratio. This makes high sensitivity measurement infeasible.
Maly, F. E., et al. (Anal. Biochem., 168 462-469(1988)) provides an immunoassay method in which a microtiter plate is used as the solid phase for objects, and the light emitted from each of the wells of the microtiter plate is detected by a two-dimensional detector. This method enables simultaneous detection of signals emitted from the individual wells. The method of Maly, however, is equivalent to a method of simultaneous point sensing for each of the wells. Maly discusses no countermeasure against the high background noise over the signals from the light-emitting reagent in the respective microtiter plate wells. Japanese Patent Application S63-201561 describes an immunoassay method that enable to obtain a two-dimensional optical image of fine particles and to counts the number of the particles. This method, however, is intended to determine the number of particles by binarizing the intensity of light signals from the fine particles, but is not directed to determine the total light intensity of an object with sufficiently high sensitivity with an improved S/N ratio.